Intergenerational effects of racism on amygdala and hippocampus resting state functional connectivity

Racism is an insidious problem with far-reaching effects on the lives of Black, Indigenous, and People of Color (BIPOC). The pervasive negative impact of racism on mental health is well documented. However, less is known about the potential downstream impacts of maternal experiences of racism on offspring neurodevelopment. This study sought to examine evidence for a biological pathway of intergenerational transmission of racism-related trauma. This study examined the effects of self-reported maternal experiences of racism on resting state functional connectivity (rsFC) in n = 25 neonates (13 female, 12 male) birthed by BIPOC mothers. Amygdala and hippocampus are brain regions involved in fear, memory, and anxiety, and are central nodes in brain networks associated with trauma-related change. We used average scores on the Experiences of Racism Scale as a continuous, voxel-wise regressor in seed-based, whole-brain connectivity analysis of anatomically defined amygdala and hippocampus seed regions of interest. All analyses controlled for infant sex and gestational age at the 2-week scanning session. More maternal racism-related experiences were associated with (1) stronger right amygdala rsFC with visual cortex and thalamus; and (2) stronger hippocampus rsFC with visual cortex and a temporo-parietal network, in neonates. The results of this research have implications for understanding how maternal experiences of racism may alter neurodevelopment, and for related social policy.


Participants
Data came from the Brain and Early Experience Study, a prospective longitudinal study investigating the effects of early experience on child development (Mills-Koonce et al., 2022).Pregnant women who resided within a 50-mile radius of UNC-Chapel Hill were identified from medical records, and through flyers, emails, and online advertisements.Potential participants were excluded if they were younger than 18 years of age, did not speak primarily English at home and/or were not experiencing a singleton pregnancy.The sample in this study represents a subsample from a larger study that was examining home and environmental factors that influence the emergence of executive function.Language interactions were one of the focal variables of the larger study and thus, as a first step, we could not include bi-or tri-lingual language learners due to the measurement programs and statistical power needed for the primary study questions.
Following birth, infants were considered as participants and mother-infant dyads were officially enrolled into the study if the infant was born at 36 weeks and 4 days gestational age or older, 5.5 pounds or heavier, not medically fragile, and did not have any medical devices implanted that would preclude infants' ability to participate in the magnetic resonance imaging (MRI) scan.If the infant did not meet eligibility criteria at birth, the dyad was not enrolled in the study.A total of 203 mother-infant dyads were officially enrolled in the study, of whom n = 81 identified as BIPOC for inclusion in the current study, and n = 79 completed the Experiences of Racism Scale (REQ).
Due to in-person data collection restrictions due to the COVID19 pandemic, the current sample represents a sub-sample of the larger BEE Study.Two participants did not complete the second data collection session (neonatal MRI visit), and 22 participants completed the session remotely, thus 55 participants had an MRI scan.Of those who received an MRI scan, 22 participants were missing resting state fMRI data due to technical issues, including waking prior to the scan (the resting state fMRI was the last sequence collected in the imaging series); and 8 participants were excluded from analysis due to excessive motion during the MRI scan (described below), resulting in n = 25 participants in analyses reported here.

Data collection
Pregnant participants completed a lab-based prenatal data collection visit during which they answered questionnaires, including demographics, income-to-needs ratio (INR), and the BSI 45 , among other measures collected to address aims of the larger project in which the current study was embedded 46 .When infants were approximately 2 weeks old participants completed a second study visit consisting of a neonatal MRI scan.At the end of each visit, mother-infant dyads were compensated with up to $125 in the form of a gift card, and a small gift of diapers and baby shampoo.All data included herein was collected prior to the pandemic-related shutdown in March 2020.changing "African American" to "race".For example, instead of "because you are African American", the adapted scale would read "because of your race".The REQ is a standardized assessment that determines how often an individual has experienced different forms of racial discrimination over the past 5 years.It consists of 13 items, such as "How often have the police hassled you just because of your race?" that are rated on a 4-point scale (0 = Never, 1 = Once or Twice, 2 = A few times, and 3 = Several times).The first 11 items refer to direct, personal experiences with racism, and the last 2 items refer to racial discrimination experienced by the person's friends and family based on their race.REQ items were summed and divided by the number of items (13) to calculate an average score.Higher scores indicate a higher number of racist experiences (α = 0.87 in the current sample; minimum = 0; maximum = 3).REQ scores were transformed with a natural log to correct for a highly skewed distribution, though results were consistent when using non-transformed REQ scores.

MRI acquisition
Images were acquired on a Siemens Prisma 3T scanner with a 32-channel head coil.Anatomical images consisted of a high-resolution 3D

MRI processing
Functional images were processed with Analysis of Functional NeuroImages (AFNI) 47 version 17.3, and the FMRIB Software Library (FSL), version 6.0.4B-0 and fieldmap correction was performed using FSL's topup 48,49 .Subsequent analysis steps performed in AFNI included: removal of the first 10 volumes; motion correction with 3dvolreg; bias field correction with 3dUnifize 50 ; registration of the subject's functional data to their anatomical image with 3dAllineate 51 .A 12-degree of freedom affine transformation was followed by nonlinear transformation using 3dNwarpApply to register each subject's functional data to University of North Carolina (UNC) 0-1-2 Infant Atlases 52 , neonate space.Images were segmented into white matter, grey matter, and cerebrospinal fluid (CSF) with FSL's FMRIB Automated Segmentation Tool (FAST) for use as masks that were eroded using a 3 × 3x3 voxel kernel and then used to generate ROI-averaged time series, with white matter and CSF time-series serving as nuisance regressors (along with their derivatives, the 6 motion regressors, global signal and its derivative) with 3dTproject.Time points where the volume-to-volume motion (Euclidean norm of the temporal differences of the 6 realignment parameters) exceeded 0.2 mm were censored during nuisance regression and replaced by interpolating from neighboring low-motion time points.Images were band-pass filtered from 0.01 to 0.10 Hz and smoothed with a 6-mm full-width half-maximum Gaussian kernel.We extracted the time-series from right and left amygdala and hippocampus seeds based on the UNC 0-1-2 Infant Atlases 52 , neonate parcellation and concatenated across the two scan runs' timeseries.We regressed each time-series (separately for each seed ROI) back onto each participant's data using AFNI's 3dDeconvolve, which also censored high-motion time-points (greater than 0.2 mm framewise displacement) 53 .Participants (n = 8) were excluded from analysis if they had less than 5 min of data due to more than 40% of data points censored for motion.Voxel-wise, whole-brain connectivity was assessed based on the Fisher-Z transformed correlation between the seed and every other voxel in the brain.

Statistical analysis
We examined the contrast for the main effect of experiences of racism on seed-based connectivity using REQ scores as a continuous, voxel-wise regressor.All voxelwise analyses were thresholded at p < 0.05 controlling for family-wise error using threshold-free cluster enhancement with FSL's Randomise 54 , and controlled for infant sex and gestational age at the 2-week MRI visit.Code for data processing and analysis can be found at the following location: https:// github.com/ KralT RA/ BEE-resti ng-fMRI.

Self-reported experiences of racism (REQ)
Participants reported an average of 8.2 experiences of racism, with a range from 0 to 28 total experiences (Fig. 1).There was no relationship between REQ scores with infant sex (p = 0.89, b = 0.04, t( 23

Resting state functional brain connectivity
Cluster details for significant results are presented in Table 2. Results are consistent when adding an additional covariate to control for motion, and the number of time-points censored for motion was unrelated to the independent variables (ps > 0.10).Statistical maps are available at the Open Science Framework at the following web address, labeled with the manuscript title: https:// osf.io/ wunrm/.www.nature.com/scientificreports/Amygdala connectivity More maternal experiences of racism were associated with stronger right amygdala rsFC, with clusters in primary visual cortex and thalamus (Fig. 2) in a whole brain, voxel-wise analysis (p < 0.05, corrected).There was no significant relationship between REQ scores and left amygdala resting state connectivity, nor between INR, PSS, or BSI scores and either amygdala seed (p > 0.05), though the effects of REQ on amygdala rsFC were not   www.nature.com/scientificreports/significant when controlling for either INR or BSI scores.The effect of REQ on right amygdala rsFC remained significant when controlling for general stress with the PSS.

Hippocampus connectivity
More maternal experiences of racism were positively associated with stronger left hippocampus rsFC with clusters in primary visual cortex (calcarine), middle and superior parietal cortex, and superior temporal cortex (Fig. 3a); and with stronger right hippocampus rsFC with clusters in superior temporal cortex, precuneus, and left parahippocampus (Fig. 3b), in a wholebrain, voxel-wise analysis (p < 0.05, corrected).The hippocampus regions of interest are depicted in blue (Fig. 3c).There was no relationship between INR, PSS, or BSI scores and hippocampus rsFC in this sample, though the effects of REQ on hippocampus rsFC were not significant when controlling for either INR or BSI scores.The effects of REQ on hippocampus rsFC remained significant when controlling for general stress on the PSS.

Discussion
This study found preliminary evidence for intergenerational effects of the experience of racism on neonates' functional brain connectivity-in networks associated with vigilance and emotional memory [55][56][57][58] .The more experiences of racism that BIPOC mothers reported, the stronger the neonatal functional connectivity of the amygdala and hippocampus to the primary visual cortex, among other regions.The current findings provide further evidence linking maternal health and well-being during pregnancy to neonatal outcomes.The results may suggest that maternal experiences of racism influence the infant brain to allow for adaptive coping to a threatening context, though such adaptations may become detrimental as individuals transition between different contexts, with differing needs for vigilance to threat.More likely, the impact of maternal experiences of racism on infant brain development is a maladaptive consequence, similar to the direct experience of trauma during childhood, which has known long-term, adverse outcomes for health and development [59][60][61] .While research is scant regarding the developmental implications of alterations in neonatal amygdala-visual cortex rsFC, prior research has found that stronger neonatal amygdala rsFC with insula was associated with higher fearfulness at 6 months old 62 , and with depressive symptoms at 2 years old 26 , consistent with the known role of these neural circuits with anxiety and fear in adults 24,63 , and in older children with fear-based anxiety disorders 64 .Thus, we would hypothesize that increased neonatal amygdala-visual cortex rsFC may predispose individuals to higher vigilance in childhood, given the association of amygdala-visual cortex connectivity with vigilance, described above.Future research is needed to examine this hypothesis and further characterize the developmental implications of altered neonatal amygdala connectivity.Similarly, while few studies have examined neonatal rsFC in relation to maternal experiences of stress, our findings of stronger hippocampal-temporal rsFC are consistent with a recent study showing that higher maternal distress was correlated with stronger connectivity between the hippocampus and the temporal lobe in neonates, across multiple measures of distress (i.e., cortisol, perceived stress, pregnancy-related distress and depression) 43 .Suggesting that alterations in connectivity between www.nature.com/scientificreports/these brain regions is shared across various measures and forms of distress, including experiences of racism.
Other studies of prenatal stress suggest that the impact on the developing brain endures into childhood and likely beyond 65 , potentially increasing the risk for behavioral or psychiatric conditions later in development 66 .These specific links have yet to be tested directly in relation to maternal experiences of racial discrimination.
Understanding the parallels between racism and trauma, and forms of psychopathology like PTSD, has the potential to inform both preventative and secondary interventions for BIPOC individuals and communities aimed at negating the harmful effects of racism.Such efforts are critical to reduce individual suffering, yet they are insufficient to address the root cause of racism.If experiences of racism lead to heightened glucocorticoids in utero, e.g., similar to other forms of stress, then reducing racism-related stress and trauma for BIPOC mothers, is critical to ensuring health and flourishing in neonates.To intervene effectively, it is also vital to recognize the unique context of trauma induced by experiences of racism [67][68][69][70] , including its ongoing nature and pervasiveness, in the lives of BIPOC individuals.
This study was limited by a small sample size, which was primarily comprised of individuals who identified as Black, and future research should seek to replicate and extend these findings with a well-powered study, and to examine subsequent behavioral outcomes in infants and children.It is promising that the results seen here mirror what was previously shown in predominantly Hispanic mothers and their neonates' , whereby discrimination was associated with stronger amygdala rsFC to fusiform-another brain region involved in visual processing 14 .This study was also limited by the self-report modality of the REQ, which may be impacted by reporter and recall biases, as with all self-report measures.Six of the 25 participants reported zero experiences of racism (within the past 5 years), and it is unknown whether zero reported experiences of racism indicated a lack of occurrence, under-reporting (possibly due to the traumatic and stigmatic nature of the experience), or other phenomena.
Importantly, the results need to be replicated in future research powered to control for, and examine potential moderation by, factors such as poverty, stress and other forms of trauma.While we failed to find significant effects of INR and BSI on amygdala or hippocampus rsFC, these variables likely have overlapping variance with the REQ in its relationship to neonatal rsFC that the present study was underpowered to parse.Conversely, the effect of REQ was maintained when controlling for general stress assessed from the PSS, and the prior research in Hispanic women found evidence that discrimination is a distinct factor from other forms of stress 14 .A larger body of research indicates that stress and experiences of racism are independently associated with health outcomes, despite their known associations with each other and complex interactions in relation to some healthrelated variables 3 .Finally, the mechanism(s) by which racism-related stress and trauma in mothers may lead to neurodevelopmental differences in neonates warrants exploration and additional, hypothesis-driven research.Heightened glucocorticoids in utero provide one promising avenue for research 71 , and future studies should examine the interaction of changes in glucocorticoids and dynamics of trauma-related brain networks, such as the salience network.Animal studies examining the intergenerational transmission of stress-related experiences offer additional potential mechanisms.These studies show that both maternal and paternal experiences of stress alter DNA methylation and gene expression in genes that regulate maturation and arborization of neurons, as well as synaptic plasticity, which then impacts the cytoarchitectonics and connectivity of the developing offspring brain 72,73 .
In addition to the contributions of this study to developmental science, the results provide evidence for the critical need to address racism and provide support and resources for BIPOC communities 74 , and this work provides initial evidence that these impacts may extend beyond the mother, with downstream intergenerational effects that are already present in the neonatal brain, just after birth.In addition to addressing the negative impacts of racism on BIPOC individuals, it is critical to address racism directly, as a problem of White supremacist culture that is present across all socioeconomic strata and institutions of our society [75][76][77][78][79] .Future research should aim to replicate the current findings, and extend this work to examine the underlying biological mechanisms for intergenerational effects of prenatal stress, racism, and trauma, and to build off the impactful work of BIPOC scholars to test the efficacy of interventions to heal trauma from racism, e.g., 80-85 .

Figure 2 .
Figure 2. Higher right amygdala rsFC was associated with more experiences of racism.The peak of the visual cortex cluster (lingual gyrus) is overlaid in (a), and the peak of the thalamus cluster is overlaid in (b), both in teal circles (p < 0.05, maximum t-value = 5.1).Left and right amygdala seeds are depicted in light and dark blue, respectively (inset).

Figure 3 .
Figure 3. Increased hippocampus resting state functional connectivity (rsFC) was associated with more experiences of racism.The association of experiences of racism with (a) left hippocampus rsFC, and (b) right hippocampus rsFC.The peaks of the largest clusters are overlaid in teal circles (p < 0.05, maximum t-value = 5.9).Left and right hippocampus seeds are depicted in light and dark blue, respectively (c).

Table 2 .
Detailed information for significant clusters sized 50 voxels or larger.Coordinates and details of significant clusters (p < 0.05).*Cluster details for significant results at p < 0.02.